1. Field of the Invention
The invention relates to a novel system, devices and method for the enzymatic assay and detection of biochemical analytes. For convenience, the novel system, devices and method will often be referred to in a generic manner, as "systems". In the system of the invention, an analyte is reacted with an electron transferase enzyme, reducing the enzyme. The reduced enzyme is colorimetrically assayed as a means of determining the concentration or the presence (or absence) of the analyte.
The invention has several embodiments which will be described hereinafter. In one embodiment, the invention relates to systems which take an analog analyte concentration input and convert it to an analog colorimetric output signal. In these systems, there is a linear relationship between the analog analyte concentration input and the analog colorimetric signal.
In another embodiment, the invention relates to a system which takes an analog analyte concentration input and converts it to a digital colorimetric output signal. In this system, there is a digital colorimetric "off-on" signal corresponding to a sought level of the analog analyte concentration input, i.e., an analog to digital system. When the analyte is below the preselected threshold concentration, no visible color change occurs; when the analyte is at or above the preselected threshold concentration, a visible color change occurs. Therefore, in the analog to digital system, there can be determined the absence or presence of the analyte in the concentration to be determined.
While there are numerous novel and unobvious embodiments of the invention (which will be described in detail hereinafter), it can be noted at the outset, that insofar as could be determined from the search of the prior art, there appears to be no system that is operative or is based on an electron transferase enzyme which is capable of analog to digital or, alternatively, analog to analog inputs and outputs.
For convenience, "analog to digital" shall be abbreviated herein as "a/d" and "analog to analog" as "a/a". Other abbreviations will be explained hereinafter.
The measurement of concentration of biochemical analytes has many applications and uses in biomedical, medical, diagnostic, industrial (e.g. genetic engineering) and numerous other fields of art as will become readily apparent to one of average skill-in-the-art to which the invention pertains. The invention provides a test of extreme or high sensitivity and accuracy and with great convenience.
The known and useable prior art methods for enzymatic analysis of the concentration of biological analytes use oxidase enzyme based aerobic reactions which proceed as follows: EQU Substrate+O.sub.2 .fwdarw.Oxidized Substrate+H.sub.2 O.sub.2
As demonstrated in the above reaction, the enzyme oxidizes the substrate by removing two electrons to form an oxidized substrate. These two electrons are then transferred to molecular oxygen to form hydrogen peroxide. In the scientific and patent literature, enzymes that catalyze this reaction are known as "oxidases" and the presence of this activity in a process is known as "oxidase activity".
In common practice, the hydrogen peroxide produced in this reaction is then reacted with an electron donating chromogen, for example, 4-aminoantipyrine, in the presence of peroxidase enzyme to form a dye. The amount of dye produced is then used as a measure of the amount of substrate that is oxidized.
Alternatively, this reaction may be monitored by the use of an oxygen electrode to measure oxygen consumption or the oxidized substrate can be directly measured, for example, by its absorbancy in the ultraviolet region.
A number of disadvantages exist with the prior art technology, the most serious of which is the requirement for molecular oxygen in the reaction. The concentration of dissolved oxygen in air saturated water is approximately 0.24 mM. This concentration is far below the concentration in bodily fluids of many medically important biological molecules. For example, alcohol at 0.1% is at a concentration of 21.7 mM, cholesterol at 200 mg/dl is approximately 5 mM, the physiological concentration of glucose is 5 mM and concentrations of up to 50 mM are encountered in diabetic disease. Therefore, in any device that quantitatively oxidizes a biological molecule, the bodily fluid must be diluted to at least less than the concentration of oxygen in a bodily fluid, i.e., about 0.24 mM, before being measured. Without such dilution, a false reading would be obtained indicative of the concentration of oxygen, not of the medically important biological molecule.
Another reason leads to the requirement for dilution of the biological fluid when the oxidase reaction as is known in the prior art, is coupled to dye production. One molecule of hydrogen peroxide is produced for every molecule of substrate oxidized, and one molecule of dye is produced for every molecule of hydrogen peroxide.
Therefore, in a system that substantially completely oxidizes the substrate, the dye concentration would be at or greater than 5 mM for the medically important biological molecules listed above. Dye-dye interactions will occur at these high concentrations, and the amount of color in the solution will not be linearly proportional to the concentration of dye. Therefore, any device that measures the amount of color in the solution would not be able to determine the concentration of substrate that was oxidized. Therefore, in the prior art, the two above-discussed problems required that the analyte be diluted prior to use. There are other drawbacks to the methods and systems of the prior art.
In view of the problems of the prior art, there exists a real need for a system, methods, and devices for the enzymatic assay and detection of biochemical analytes which do not have the aforementioned disadvantages.
In view of the problems associated with the oxygen concentration in biological fluids described above, there exists a particularly serious need for an accurate analytical system in which oxygen presents no limiting factor in the reaction. As described in greater detail hereinafter, the system (and the process) of the invention is independent of oxygen in the analyte; i.e., the system of the invention is operative regardless of whether or not oxygen is present. Indeed, the system is capable of operating in the absence of oxygen, but oxygen can also be present. This is one of the distinctive features of the invention. Oxygen is not used in the reaction of the invention, as further described herein.